The most common form of familial ALS (FALS) is due to mutations in the Cu.Zn superoxide dismutase (SOD1) gene. SOD1-FALS is caused by a "toxic gain of function". Mitochondrial dysfunction has been demonstrated in SOD1-FALS models, and a growing body of evidence suggests that it may play a role in the pathogenesis of the disease. Although the majority of SOD1 resides in the cytosol, a significant proportion of SOD1 is localized in the mitochondria. The pathological role of mitochondrial SOD1 in FALS remains to be clarified. We propose to investigate the involvement of mitochondrial SOD1 in mitochondrial dysfunction and its role in the pathogenesis of SOD1-FALS. 1) Mitochondrial dysfunction, potentially resulting in energy depletion and apoptosis, has been observed in transgenic mice expressing mutant SOD1. To determine if mutant SOD1 localized in mitochondria causes mitochondrial dysfunction we will study neuronal cells expressing mutant or wild type SOD1 selectively targeted to mitochondria by specific cleavable protein targeting sequences. 2) To determine whether mitochondrial dysfunction associated with FALS is directly caused by mutant SOD1 in mitochondria and whether the selective expression of mutant SOD1 in mitochondria results in a pathological phenotype in vivo we will generate and study transgenic mice expressing mutant or wild type SOD1 targeted to mitochondria by specific cleavable protein targeting sequences. 3) Very little is known about the mechanisms underlying import of SOD1 in mammalian mitochondria. We will investigate: A) the molecular mechanisms involved in SOD1 mitochondrial import in affected and unaffected tissues;B) which protein domains are implicated in SOD1 mitochondrial import;C) the impact of SOD1 post-translational modifications and conformation on mitochondrial import. 4) Preliminary results suggest that SOD1 interacts with other mitochondrial proteins. A) Proteins with mitochondrial isoforms have been shown to interact with mutant but not wild type SOD1 in a yeast two- hybrid system. We will study these interactions in mammalian cells. B) We will search for novel protein interactions between SOD1 and mitochondrial proteins.